The present invention relates generally to inhibition of protein activity, and in particular kinase activity, and compounds and assays for measuring such inhibition useful for identifying novel inhibitors of kinas activity. In particular, the present invention relates Nef protein and inhibition of its interaction and complexes comprising Nef, with other cellular components as described herein and known in the art.
The information provided herein is intended solely to assist the understanding of the reader. None of the information provided nor references cited is admitted to be prior art to the present invention. Each of the references cited herein is incorporated in its entirety and for all purposes.
The HIV-1 nef gene encodes a small myristoylated accessory protein Nef, having NCBI Locus CAA41585 (SEQ ID NO: 1), which protein is required for optimal viral replication and AIDS pathogenesis (Fackler, O. T. and Baur, A. S., 2002, Immunity. 16:493-497; Geyer, M., Fackler, O. T. and Peterlin, B. M., 2001, EMBO Rep. 2:580-585). It is understood that the term “NCBI Locus” refers to a unique alphanumeric identifier given to a sequence deposited with the National Center for Biotechnology Information. Deletion of nef from the HIV-related simian immunodeficiency virus (SIV) prevents AIDS-like disease progression in rhesus macaques (Kestler, H. W., III, Ringler, D. J., Mori, K., Panicali, D I., Sehgal, P. K., Daniel, M. D. and Desrosiers, R. C., 1991, Cell 65:651-662). In addition, expression of the nef gene alone is sufficient to induce an AIDS-like syndrome in transgenic mice very similar to that observed upon expression of the complete HIV-1 provirus therein (Hanna, Z., Kay, D. G., Cool, M., Jothy, S., Rebai, N. and Jolicoeur, P., 1998, J. Virol. 72:121-132; Hanna, Z., Kay, D. G., Rebai, N., Guimond, A., Jothy, S. and Jolicoeur, P., 1998, Cell 95:163-175). In humans, nef sequence variability and function correlate with HIV disease progression over the course of infection (Carl, S., Greenough, T. C., Krumbiegel, M., Greenberg, M., Skowronski, J., Sullivan, J. L. and Kirchhoff, F., 2001, J. Virol. 75:3657-3665; Kirchhoff, F., Easterbrook, P. J., Douglas, N., Troop, M., Greenough, T. C., Weber, J., Carl, S., Sullivan, J. L. and Daniels, R. S., 1999, J. Virol. 73:5497-5508). Indeed, long-term non-progressive HIV infection has been associated with nef-defective strains of HIV in some cases (Deacon, N. J., Tsykin, A., Solomon, A., Smith, K., Ludford-Menting, M., Hooker, D. J., McPhee, D. A., Greenway, A. L., Ellett, A., Chatfield, C., 1995, Science 270:988-991; Kirchhoff, F., Greenough, T. C., Brettler, D. B., Sullivan, J. L. and Desrosiers, R. C., 1995, N. Engl. J. Med. 332:228-232). Thus, several studies identify the HIV-1 Nef protein as a key molecular determinant of AIDS and related conditions (e.g., AIDS-related syndrome).
Because Nef lacks any known intrinsic enzymatic or biochemical function, it is believed that Nef exploits multiple host cell signaling pathways to optimize conditions for viral replication and AIDS progression (Fackler, O. T. and Baur, A. S., Id.; Greenway, A. L., Holloway, G., McPhee, D. A., Ellis, P., Cornall, A. and Lidman, M., 2003, J. Biosci. 28:323-335; Joseph, A. M., Kumar, M. and Mitra, D., 2005, Curr. HIV. Res. 3:87-94; Piguet, V. & Trono, D., 1999, Reviews in Medical Virology, 9:111-120; Renkema, G. H. & Saksela, K., 2000, Front Biosci. 5:D268-D283). Indeed, Nef binding influences several classes of signaling molecules, including immune receptors, trafficking proteins, guanine nucleotide exchange factors, and protein kinases (Arold, S. T. & Baur, A. S., 2001, Trends Biochem. Sci. 26:356-363; Geyer, M., et al., 2001, Id.). These Nef-mediated interactions enhance viral replication and contribute to immune evasion as well as survival of infected cells (Briggs, S. D. et al., 2001, J. Biol. Chem. 276:25605-25611; Choi, H.-J. & Smithgall, T. E., 2004, Biol. Chem. 279:51668-51696; Geleziunas, R., Xu, W., Takeda, K., Ichijo, H. & Greene, W. C., 2001, Nature 410:834-838).
The prior art identifies the Src family kinases (SFKs), a group of non-receptor protein-tyrosine kinases that control cell growth, differentiation, and survival (Parsons, S. J. & Parsons, J. T., 2004, Oncogene 23:7906-7909; Thomas, S. M. & Brugge, J. S., 1997, Annu. Rev. Cell Dev. Biol. 13:513-609), as key molecular targets for Nef (Greenway, A. L., Holloway, G., McPhee, D. A., Ellis, P., Cornall, A. and Lidman, M., 2003, J. Biosci. 28:323-335; Renkema, G. H. and Saksela, K., 2000, Front Biosci. 5:D268-D283). Accordingly, members of the Src family of non-receptor protein-tyrosine kinases represent an important class of Nef target proteins. Nef binds to the Src homology 3 (SH3) domains from the Src family members Fyn, Hck, Lck, Lyn and c-Src, all of which are expressed in HIV-1 target cells (Saksela, K., Cheng, G. & Baltimore, D., 1995, EMBO J. 14:484-491; Arold, S. et al. 1997, Structure 5:1361-1372; Choi, H. J. & Smithgall, T. E., 2004, J. Mol. Biol. 343:1255-1268; Arold, S. et al., 1998, Biochemistry 37:14683-14691). It is believed that Nef activates c-Src and Lyn through a similar mechanism, suggesting that Nef-mediated Src family kinase activation is a common feature of HIV-infected cells (Trible, R. P., Emert-Sedlak, L. & Smithgall, T. E., 2006, J. Biol. Chem. 281:27029-27038.
The SFK Hck is a Src family member expressed in macrophages that binds strongly to Nef via an SH3-mediated interaction (Arold, S., et al., Id.; Lee, C. H., Leung, B., Lemmon, M. A., Zhong, J., Cowburn, D., Kuriyan, J. and Saksela, K., 1995, EMBO J. 14:5006-5015). Nef induces constitutive activation of Hck through a mechanism that involves displacement of the SH3 domains negative regulatory interaction with the catalytic domain (Moarefi, I. et al., 1997, Nature 385:650-653; Briggs, S. D., Sharkey, M., Stevenson, M. & Smithgall, T. E., 1997, J. Biol. Chem. 272:17899-17902). Nef binding leads to constitutive Hck activation (Briggs, S. D., et al., Id.; Lerner, E. C. & Smithgall, T. E., 2002, Nat. Struct. Biol. 9:365-369; Moarefi, I., LaFevre-Bernt, M., Sicheri, F., Huse, M., Lee, C. H., Kuriyan, J. and Miller, W. T., 1997, Nature 385:650-653; Trible, R. P., Emert-Sedlak, L. and Smithgall, T. E., 2006, J. Biol. Chem. 281:27029-27038), which may be important for macrophage survival (Briggs, S. D., Scholtz, B., Jacque, J. M., Swingler, S., Stevenson, M. and Smithgall, T. E., 2001, J. Biol. Chem. 276:25605-25611; Choi, H. J. & Smithgall, T. E., 2004, J. Biol. Chem. 279:51688-51696) and productive infection by M-tropic HIV (Komuro, I., Yokota, Y., Yasuda, S., Iwamoto, A. and Kagawa, K. S., 2003, Exp. Med. 198:443-453). Strikingly, transgenic mice expressing a Nef mutant lacking a highly conserved PxxPxR motif essential for activation of Hck and other SFKs showed no evidence of AIDS-like disease (Hanna, Z., Weng, X., Kay, D. G., Poudrier, J., Lowell, C. and Jolicoeur, P., 2001, J. Viral. 75:9378-9392). Furthermore, when the Nef-transgenic mice were crossed into a hck-null background, appearance of AIDS-like phenotype was delayed and mortality was reduced (Hanna, Z., et al., Id.). While not dictating adherence to a particular theory, these observations support an essential role for Nef:SFK interactions in AIDS pathogenesis and suggest that small molecules targeted to Nef:SFK complexes may represent new leads for anti-HIV therapy. It is understood that the terms “Nef:SFK,” “Nef:SFK complex” and the like refer to the complex formed between Nef and an SFK, for example, Nef:Hck.
To reiterate, lack of a catalytic function makes analyses of the interaction of Nef with small molecule inhibitors by a variety of approaches, such as high-throughput screening (HTS) approaches, problematic. Accordingly, the present invention provides assays suitable to identify inhibitors of Nef:SFK (e.g., Nef-Hck) signaling despite the lack of a catalytic function for Nef.
The screening assays of the present inventionare useful for identifying inhibitors of kinase activity of Nef:SFK complex. The invention further provides compounds useful as drugs resulting from the use of such assays.